The present invention relates generally to recombinant methods and materials useful for securing the microbial expression of exogenous gene products.
Streptococcus mutans, which causes dental caries, adheres to the surface of tooth enamel through the synthesis of extracellular polysaccharide polymers from sucrose. The most important of these polysaccharides are the glucans. One type of bond contained in glucans is the .alpha.-1,6 bond (see FIG. 1) which is similar to that of the classical dextrans. A second type of bond contained in glucans is the .alpha.-1,3 bond which is less water soluble. S.mutans and other cariogenic microorganisms cause tooth decay by adhesion to the tooth surface and secretion of organic acids and other material which causes demineralization of the inorganic component of tooth structure and dissolution of the residual organic matrix.
In the past, dental plaque (which is a complex of cariogenic bacteria, insoluble glucans and other material) has been mechanically removed by brushing the teeth with a tooth brush and use of materials such as dental floss. It is significant, however, that bacteria sorb rapidly to the enamel surface within minutes after the teeth are vigorously cleaned and that macroscopically visible colonies will then appear within one or two days. In addition, it is extremely difficult to ensure thorough cleaning of the teeth as dental plaque will often remain intact in areas difficult to reach with a tooth brush or dental floss.
Recently, dentifrices have been developed which provide for the enzymatic decomposition of the .alpha.-1,3 and .alpha.-1,6 glucosidic bonds holding together insoluble glucan polymers. Such dentifrices typically comprise .alpha.-1,6-glucan 6-glucanohydrolase (.alpha.-1,6 glucanase or dextranase and hereinafter referred to as dextranase) or .alpha.-1,3-glucan 3-glucanohydrolase (.alpha.-1,3 glucanase or mutanase and hereinafter referred to as .alpha.-1,3 glucanase). Simonson, et al., U.S. Pat. No. 4,328,313 and Guggenheim, et al., U.S. Pat. No. 4,353,891 each disclose methods for production of plaque dispersing .alpha.-1,3 glucanase enzyme. Shimada, et al., U.S. Pat. No. 4,438,093 discloses an oral composition for the prevention and suppression of oral diseases comprising both .alpha.-1,3 glucanse and dextranase in a pharmaceutically acceptable carrier. The application of dentifrices containing enzymes for the disintegration of such insoluble glucan is not expected to have significant effects in decomposing dental plaque lasting beyond the period of brushing because most of the enzymatic components of the dentifrices tend to be lost during the rinsing which typically follows brushing.
Prior to the filing of parent application Ser. No. 06/840,940 herein, there had been no reports of the use of recombinant methods in the cloning and isolation of genes coding for .alpha.-1,3 glucanase or dextranase enzymes. Since that time, the cloning and expression in E. coli of a gene encoding the dextranase product (EC 3.2.1.70) of Bacteroides oralis Ig4a has been reported. See, Derwent, Biotechnology Abstracts 88-00247 and 88-01356 (1988).
From the above description of the state of the art it is apparent that there exists a need in the art for improved methods and materials providing glucanase activity in the human oral cavity. Such methods and materials should preferably provide consistent long lasting glucanase activity and should preferably not require frequent application.